Spinal and epidural techniques provide surgical anesthesia as well as prolonged analgesia for post-operative pain, labor pain and chronic cancer pain. Molecular mechanisms underlying spinal action of local anesthetics and opioid analgesics are examined here by complementary biochemical and electrophysiological studies of primary sensory neurons (dorsal root ganglion, DRG) and dorsal spinal cord (DSC) cells maintained in culture. Substance P (SP), substance K (SK), and neuromedin K (NMK) are members of the tachykinin (TK) family of neuropeptides. SP, the best studied of these peptides, has been implicated as a neuromodulator in transmission from primary sensory neurons to cells in the dorsal spinal cord. A proposed mode of action of spinal opioid peptides and narcotic analgesics is via pre-synaptic inhibition of SP release from sensory neurons. We propose to examine effects of acute and chronic exposure to mu, delta, and kappa opioid agonists on electrically-evoked release of SP and SK from cultured DRG. These studies address the roles of the individual TK peptides and the mechanisms underlying tolerance and cross-tolerance to spinal narcotics. Regulation of TK biosynthesis will be examined using specific cDNA and oligonucleotide probes for RNA sequences along with immunodetection of protachykinin precursors and mature TK peptides. Calcitonin-gene related peptide (CGRP) is synthesized and released by small sensory neurons and postulated to be a neuromodulator in nociception. Sensory neurons which contain TK appear to also contain CGRP. We shall compare patterns of biosynthesis and release CGRP to those found for the TKs. Local anesthetics (LA) producing spinal anesthesia may act by inhibiting several processes, including (1) impulse conduction in dorsal roots, (2) release of neurotransmitters and neuromodulators from primary sensory afferent terminals, and (3) post-synaptic responses to transmitters in the dorsal spinal cord. Study of the relative importance of these alternatives is important to the design of more controllable or modality-specific spinal anesthetic or analgesic agents. Direct actions on calcium channels and on calcium-dependent neurosecretion from dorsal root ganglion cells will be examined both by electrophysiologic measurements and by modulation of electrically evoked TK release by specific modifiers of ion channel function. Post-synaptic ion currents and their modulation by local anesthetics will be examined under patch clamp of cultured dorsal horn neurons exposed by iontophoresis or pressure injection to excitatory neurotransmitters (glutamate) and excitatory neuromodulators (TK) and adrenergic agents. Effects of LA on synaptic transmission from DRG to DSC cell individually identified in co-culture will be examined for effects on: (1) ion currents in DRG and DSC cells, (2) evoked TK release from DRG cells, and (3) TK and CGRP biosynthesis in DRG cells. The proposed studies should provide a model system for detailed mechanistic study that will permit more rational design and testing of spinal anesthetic an analgesic agents.